The long-term objective of this work is to develop predictable and reproducible therapies for soft tissue regeneration using adipose stem cells (ASCs) formulated as 3-dimensional (3-D) multicellular aggregates. Currently available treatments are limited by risk, morbidities, and/or reliability. Cell based tissue engineering strategies are quickly emerging as a promising approach to adipose tissue replacement and regeneration, with a goal to provide platforms based on scientific principles. In addition to providing a novel approach to adipose tissue regeneration, this work will also generate in vitro and in vivo models that will be of use for understanding obesity and the treatment thereof, as well as the biology of cell niches and the role of the micro-milieu in controlling cell survival and fate. Aim 1 studies will characterize the effect of serum-free 3-D suspension culture on ASC differentiation in vitro, yielding a defined system that will enable further mechanistic studies. Aim 2 studies will characterize how 3-D suspension culture impacts ASC survival and adipogenic differentiation in vivo using a NOD SCID mouse model. Aim 3 studies will characterize the impact of recipient site (milieu) on the engraftment and differentiation of ASCs prepared and delivered as multicellular aggregates. Together, these studies will provide insights as to how culture method (i.e. 3-D suspension culture, serum-free medium) influences the biological activity and therapeutic efficacy of ASCs. PUBLIC HEALTH RELEVANCE Cell-based regenerative medicine strategies hold tremendous potential for the treatment of human disease and trauma. However, this emerging discipline is in need of techniques and methods that enable reproducible and predictable outcomes. This proposal will pursue a novel `modular'approach to soft tissue reconstruction/regeneration using prefabricated multicellular aggregates that approximate cell structures reported in the literature to be responsible for normal adipose tissue development.